Nlrp12 Mediates Adverse Neutrophil Recruitment During Influenza Virus Infection Emma E

Nlrp12 Mediates Adverse Neutrophil Recruitment during Influenza Virus Infection Emma E. Hornick, Balaji Banoth, Ann M. Miller, Zeb R. Zacharias, Nidhi Jain, Mary E. Wilson, Katherine N. This information is current as Gibson-Corley, Kevin L. Legge, Gail A. Bishop, Fayyaz S. of October 2, 2021. Sutterwala and Suzanne L. Cassel J Immunol published online 27 December 2017 http://www.jimmunol.org/content/early/2017/12/27/jimmun ol.1700999 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 27, 2017, doi:10.4049/jimmunol.1700999 The Journal of Immunology

Nlrp12 Mediates Adverse Neutrophil Recruitment during Inﬂuenza Virus Infection

Emma E. Hornick,* Balaji Banoth,† Ann M. Miller,* Zeb R. Zacharias,*,‡ Nidhi Jain,† Mary E. Wilson,*,x,{ Katherine N. Gibson-Corley,‡ Kevin L. Legge,*,‡,‖ Gail A. Bishop,*,x,{,‖ Fayyaz S. Sutterwala,*,†,1 and Suzanne L. Cassel*,†,1

Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutro- phils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation, however, neutrophils are necessary for optimal viral control. In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp122/2 mice are protected from lethality during IAV infection and show decreased vascular permeability, fewer pulmonary neutrophils, and a 2/2

reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12 neutrophils and Downloaded from dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp122/2 dendritic cells was not due to a difference in CXCL1 protein stability, but instead to a decrease in Cxcl1 mRNA stability. Together, these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses. The Journal of Immunology, 2018, 200: 000–000.

evere disease during influenza A virus (IAV) infection is (1, 4, 5). Subsequent studies have revealed a much more nuanced http://www.jimmunol.org/ associated with excessive cytokine production and an ex- relationship between neutrophils and the IAV-infected lung. S aggerated innate immune response, leading to substantial Neutrophils are among the first immune cells to arrive in the tissue damage and impaired respiratory function (1–3). The lungs, where they contribute to viral clearance through phagocy- heightened morbidity and mortality among otherwise healthy tosis of viral particles and IAV-infected apoptotic cells (6). Neu- adults infected with the 2009 pandemic H1N1 strain emphasized trophil depletion in mice during IAV infection results in increased the threat posed by emerging strains to which there is little or no viral titers and mortality (7, 8), however, mice deficient in key pre-existing immunity in the population. neutrophil effector molecules such as myeloperoxidase or che- The presence of elevated neutrophil chemoattractants and moattractants such as CXCL2 have less severe disease (9, 10). A massive neutrophil infiltration of the lungs in lethal cases of IAV recent study provided an excellent framework for understanding by guest on October 2, 2021 suggested a detrimental role for neutrophils during IAV infection these dissonant results, finding that enhancement of inflammatory signaling networks driven largely by neutrophils is an early pre- dictor of lethality (7, 11). Key effector molecules downstream of *Interdisciplinary Program in Immunology, University of Iowa Carver College of Medicine, Iowa City, IA 52242; †Department of Medicine, Cedars-Sinai Medical activation of these networks include neutrophil chemoattractants Center, Los Angeles, CA 90048; ‡Department of Pathology, University of Iowa (CXCL1, 2, and 5) and their receptor (CXCR2). Partial depletion x Carver College of Medicine, Iowa City, IA 52242; Department of Internal Medicine, of neutrophils reduced mortality during lethal infection, support- University of Iowa Carver College of Medicine, Iowa City, IA 52242; {Veterans Affairs Medical Center, Iowa City, IA 52246; and ‖Department of Microbiology ing the notion that control of early inflammatory responses is key and Immunology, University of Iowa Carver College of Medicine, Iowa City, to survival in high-dose infections (11). IA 52242 Nlrp12 is a member of the nucleotide binding domain and 1 F.S.S. and S.L.C. contributed equally to this work. leucine rich repeat containing receptor family, which has been ORCIDs: 0000-0002-7467-2198 (B.B.); 0000-0001-9157-3469 (Z.R.Z.); 0000-0001- implicated in regulation of proinflammatory signaling in the 6723-4029 (N.J.); 0000-0002-1291-5078 (G.A.B.). context of bacterial infections, tumorigenesis, and autoimmunity Received for publication July 12, 2017. Accepted for publication November 28, (12–14). We and others have recently shown that Nlrp12-deficient 2017. cells produce decreased CXCL1, a potent neutrophil chemo- This work was supported by National Institutes of Health grants R01 AI118719 (to F.S.S.), R01 AI104706 (to S.L.C.), and an American Lung Association/ attractant, during bacterial infections (13, 15). Given the complex American Academy of Allergy, Asthma & Immunology Foundation grant (to S.L.C.). role of neutrophils during IAV infection, we sought to determine E.E.H. was supported by National Institutes of Health Grant T32 AI007485 (to G.A.B.). whether Nlrp12 influenced host susceptibility to IAVinfection. We This article is based upon work supported in part by facilities and equipment provided by 2/2 the Department of Veterans Affairs, Veterans Health Administration, Office of Research report that Nlrp12 mice have significantly improved survival and Development. following IAV infection in comparison with wild-type (WT) mice. 2 2 Address correspondence and reprint requests to Suzanne L. Cassel, Cedars-Sinai Nlrp12 / mice maintained their ability to control infection and Medical Center, 127 S. San Vicente Boulevard, AHSP, Room A9402, Los Angeles, have decreased CXCL1-driven pulmonary vascular permeability CA 90048. E-mail address: [email protected] and pulmonary neutrophil recruitment. The online version of this article contains supplemental material. Abbreviations used in this article: ActD, actinomycin D; ARE, AU-rich element; BMDC, bone marrow–derived dendritic cell; BMM, bone marrow–derived macro- Materials and Methods phage; CT, cycle threshold; IAV, influenza A virus; PRR, pattern recognition recep- Mice tor; qRT-PCR, quantitative RT-PCR; TTP, tristetraprolin; WT, wild type. The generation of Nlrp122/2 mice has been described elsewhere (16). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Mice were backcrossed to C57BL/6N or BALB/cJ mice for at least nine

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700999 2 Nlrp12 ACTIVATION IS DETRIMENTAL IN INFLUENZA generations and maintained in a specific pathogen-free facility. C57BL/6N Microvascular permeability mice were purchased from the Charles River Laboratories and used as WT controls unless otherwise stated; BALB/cJ and C57BL/6J mice We assessed microvascular permeability by extravasation of Evans blue dye were purchased from the Jackson Laboratory. Both male and female mice as previously described (19). Briefly, mice were injected i.v. with 20 mg/kg 6–10 wk of age were used, however, mice were sex, age, and weight matched Evans blue dye. Then 1 h later, lungs were perfused with PBS, weighed, for individual experiments. The Institutional Animal Care and Use Com- and homogenized. Evans blue dye in lung homogenates was extracted with mittee at the University of Iowa approved all protocols used in this study. formamide (Sigma-Aldrich) at 60˚C for 18 h. Absorbance of lung homogenate supernatants at 620 and 740 nm was measured to calculate Virus and in vivo infection micrograms Evans blue dye per gram of lung tissue. Mouse-adapted IAV strain A/PR/8/34 was propagated as previously de- In vitro infection and stimulation of bone marrow–derived cells scribed (17). Mice were anesthetized with ketamine and xylazine and infected intranasally with virus diluted in 50 ml sterile DMEM. Weight was Bone marrow–derived dendritic cells (BMDCs) and bone marrow–derived monitored daily and mice were euthanized upon losing 30% of their macrophages (BMMs) were generated as described (13, 20). Bone marrow starting weight. For CXCL1 blocking experiments, mice were adminis- neutrophils were purified by negative selection with an EasySep Mouse tered 5 mg anti-CXCL1 (MAB453, clone 48415; R&D Systems) or isotype Neutrophil Enrichment Kit (Stemcell Technologies), following the man- control Ab i.p. immediately prior to infection, and 24 and 72 h post- ufacturer’s instructions. On day 6 of culture, 1 3 106 BMDCs were infection. infected with IAV (multiplicity of infection = 5) in serum-free infection medium for 60 min, then washed twice with PBS and incubated in complete Lung titers, sectioning, and histology medium until harvest. Alternatively, BMDCs were treated with 1 mg/ml To measure virus titers, lungs were homogenized using a Tissue-Tearor R848 (InvivoGen) in complete medium until harvest. For transcript stabil- (Biospec), then homogenates were clarified by centrifugation and imme- ity experiments, 10 mg/ml actinomycin D (ActD) (Sigma-Aldrich) was diately frozen at 280˚C. A standard plaque assay on Madin-Darby canine added to BMDCs, BMMs, or neutrophils after 3 h of 1 mg/ml R848 stimulation. For protein stability experiments, 10 mg/ml cycloheximide (Sigma- kidney cells was subsequently used to quantify infectious virus. For his- Downloaded from tology, lungs were fixed in 10% neutral buffered formalin, embedded, cut Aldrich) was added to BMDCs after 6 h of R848 stimulation. To assess at 5 mm, and stained with H&E. A single H&E-stained slide from each of neutrophil death, freshly isolated neutrophils were incubated with 30 ng/ml five separate animals per group was evaluated by a board-certified veter- PMA (Sigma-Aldrich), 4 ng/ml CXCL1 (#250-11; PeproTech) or in com- inary pathologist in a blinded fashion, and both necrosis and pulmonary plete medium until harvest. Incubation with IAV was performed as for inflammation were assessed using semiquantitative scoring. Necrosis was BMDCs (see above). defined by cellular necrosis of the bronchiolar epithelial cells and/or the Gene expression analysis pneumocytes, which was accompanied by accumulation of necrotic cellular debris in these regions. The entire slide was evaluated and included RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) per the http://www.jimmunol.org/ both the right and left lung lobes. Scores were 0 = no necrosis; 1 = rare manufacturer’s instructions. cDNA was synthesized using Superscript III (1–3) scattered areas of necrosis throughout the lung; 2 = multifocal (3–9) First Strand Synthesis SuperMix (Invitrogen). Quantitative PCR was per- scattered areas of necrosis throughout the lung; 3 = numerous (.10) formed using cDNA, primer pairs for Cxcl1, Il6, and Actb (PPM03058C, areas of necrosis throughout the lung. Inflammation was defined by the PPM03015A, and PPM02945B; Qiagen), and PerfeCta SYBR Green accumulation of inflammatory cells, primarily viable and degenerate Fastmix (Quanta Biosciences). Reactions were run in a QuantStudio 6 Flex neutrophils, within airways and/or alveolar spaces. Scores were 0 = no (Applied Biosystems) with a standard cycling protocol (90˚C 2 min; 40 inflammation; 1 = 1–10% of the pulmonary parenchyma contains in- cycles of 95˚C 15 s, 60˚C 1 min). Quantitative PCR data were analyzed flammatory cell infiltrates; 2 = 10–50% of the pulmonary parenchyma using the 22DDCt method where DD cycle threshold (Ct) = [(Ct gene of contains inflammatory cell infiltrates; 3 = .50% the pulmonary paren- interest-Ct housekeeping) stimulated 2 (Ct gene of interest-Ct house- chyma contains inflammatory cell infiltrates. keeping) unstimulated]. Actb was chosen as a housekeeping gene. by guest on October 2, 2021 ELISA Western blotting Cytokines and chemokines were quantified in cell culture supernatants and Lysates were prepared in radioimmunoprecipitation assay buffer (Cell lung homogenate supernatants using DuoSet mouse ELISA kits from R&D Signaling Technology) with 1 mM PMSF per the manufacturer’s instruc- Systems: CXCL1, CXCL2, CXCL5, IL-1b, CCL2, and CCL5; or tions. Proteins were separated on a NuPAGE gel (Invitrogen) and trans- ReadySetGo! mouse ELISA kits from eBioscience: IL-6, TNF-a, IL-10, ferred to a polyvinylidene difluoride membrane using the XCell II blotting IL-1a, IL-12p40, and IFN-g following the manufacturer’s instructions. system (Invitrogen). Membranes were blocked with 5% nonfat milk and Flow cytometry incubated with primary Ab overnight at 4˚C. Primary Abs were purchased from Cell Signaling Technologies: IRAK1 (#4504), phospho-IkBa For some experiments, intravascular staining for CD45.2 was performed as (#2859), IkBa (#4814), phospho-p38 MAPK (#4511), p38 MAPK described (18) prior to tissue harvest. Single-cell suspensions were pre- (#8690), phospho-p44/42 MAPK (ERK1/2, #4370), p44/p42 MAPK pared by pressing tissues through wire mesh screens. Live cells were (ERK1/2, #4695), NFkB2 p100/p52 (#4882), phospho–NF-kB p65 enumerated by Trypan blue exclusion. Next, 1 3 106 cells per well were (#3033), NF-kB p65 (#8242) phospho-TBK1 (#5483), TBK1 (#3504). blocked with 2% rat serum and anti-mouse CD16/32 (clone 2.4G2; Tonbo CXCL1 (PA1-29220; Thermo Fisher Scientific), GAPDH (CB1001), Biosciences) in FACS buffer for 30 min at 4˚C. Following blocking, cells and total actin Ab (MAB1501) were purchased from EMD Millipore. HuR were stained in FACS buffer with fluorochrome-conjugated Abs in the dark (sc-5261), IRAK1 (sc-5288), phospho-MAPKAPK2 (sc-293139), for 30 min at 4˚C. Cells were then fixed in FACS Lysis Buffer (BD) per the MAPKAPK2 (sc-393609), and tristetraprolin (TTP) (sc-374305) were manufacturer’s instructions and resuspended in PBS. The following purchased from Santa Cruz Biotechnology. TRAF2 (592) was purchased fluorochrome-conjugated Abs were used: CD4 (clone GK1.5), CD8a from Medical & Biological Laboratories. Following washing, membranes (53-6.7), CD11a (M17/4), CD11b (M1/70), CD45.2 (104), CD49d (R1-2), were incubated with HRP-tagged anti-mouse IgG (1706516; Bio-Rad) or CD64 (X54-5/7.1FC), CD90.2 (30-H12), Ly6G (1A8), Ly6C (HK1.4), and anti-rabbit IgG (NA934; GE Healthcare) and developed using SuperSignal I-A/I-E (M5/114.15.2) from BioLegend; CD4 (RM4-5) from eBioscience; West Pico or Femto substrate (Thermo Fisher Scientific). and CD11c (HL3) from BD Biosciences. Data were acquired on a BD LSR II and analyzed with FlowJo software (FlowJo). For detection of intra- Statistics cellular CXCL1, mice were treated with 500 mg monensin (Sigma- Data were graphed and the indicated statistical tests performed using Aldrich) i.p. 5 h prior to harvest to inhibit cytokine/chemokine secretion. GraphPad Prism software. Tissue processing and surface staining was performed as above, but 0.1 mg/ml monensin and 5 mg/ml brefeldin A (Sigma-Aldrich) was present in all solutions until fixation. Cells were fixed, permeabilized, and stained Results using a Transcription Factor Staining Kit (eBioscience) according to the manufacturer’s instructions. CXCL1 was detected by biotinylated anti- Nlrp12 deficiency improves survival following IAV infection CXCL1 (BAF453; R&D Systems) and APC-labeled streptavidin (eBio- To determine whether Nlrp12 is important for protection during a science). Live cells were sorted for gene expression analysis on a BD 2/2 FACS Aria II. Hoechst dye was added to cells prior to sorting to identify high-dose IAVinfection, we infected WT C57BL/6N and Nlrp12 live cells, which were collected and processed for gene expression analysis mice with a 4LD50 inoculum of IAV, and monitored survival and (as below) after sorting. weight loss as a measure of morbidity. Strikingly, 67% of The Journal of Immunology 3

Nlrp122/2 mice survived the infection, compared with 0% sur- in the amount of these cytokines at early or late time points in the vival in the WT group at day 14 postinfection (Fig. 1A). Apart lungs of WT and Nlrp12-deficient mice (Fig. 2A–C). In contrast, from a failure of WT mice to regain weight after day 10, no neutrophil chemoattractant CXCL1 was dramatically reduced in significant differences in weight loss were apparent (Fig. 1A). Nlrp122/2 mice compared with WT mice at day 3 but not day 7 Similar results were observed in Nlrp122/2 mice backcrossed postinfection (Fig. 2D). Neutrophil chemoattractants CXCL2 and onto a BALB/c background (Supplemental Fig. 1A). CXCL5 were not different between the two groups, nor were a T cell responses are critical for protection during a primary IAV number of other cytokines and chemokines involved in inflam- infection, and augmentation of the CD8+ T cell response during mation and recruitment of innate and adaptive immune cells high-dose infections improves survival (17). A recent study sug- (Fig. 2E, 2F, Supplemental Fig. 1D–I). Therefore, Nlrp122/2 mice gested that Nlrp122/2 CD4+ and CD8+ T cells exhibit increased have a specific reduction in pulmonary CXCL1 during the early expansion and proinflammatory cytokine production upon stimu- phase of IAV infection. 2/2 lation (12), prompting us to investigate the possibility that Nlrp12 2 2 Nlrp12 / mice have decreased pulmonary neutrophils mice had enhanced T cell responses during high-dose IAV infection. Interestingly, we found no difference in the magnitude of the IAV- Neutrophils contribute to both viral clearance and immunopa- specific CD8+ or CD4+ T cell responses (Fig. 1B, 1C, Supplemental thology during IAV infection (7). They are recruited to the lungs Fig. 1B, 1C) (21). Consistent with the importance of IAV-specific early and peak 3–5 d after lethal PR8 infection (8). Following IAV CD8+ T cells for viral clearance, we observed no difference in virus infection, we assessed inflammatory cell infiltration into the lung titers in the lungs of WT and Nlrp122/2 miceatday3or7post- by flow cytometry (Fig. 3, Supplemental Fig. 2). At day 3 postinfection (Fig. 1D). Histopathologic analysis indicated more pa- infection, we found that pulmonary neutrophils were significantly thology overall in the lungs of WT mice than Nlrp122/2 mice at day decreased, both in absolute number and percentage, in the lungs of Downloaded from 2/2 5 postinfection, with increased hemorrhage and necrotic debris in Nlrp12 mice compared with WT (Fig. 3, Supplemental Fig. airways and alveoli (Fig. 1E, 1F). 2B). Intravascular staining revealed no difference in the percentage of neutrophils present in the lung vasculature of Nlrp122/2 Nlrp12 modulates CXCL1 during IAV infection mice compared with WT (Supplemental Fig. 2B). This difference Unrestrained proinflammatory cytokine production is a hallmark of in pulmonary neutrophils was still present at days 5 and 7 post- severe disease during IAV infections in humans and mice (2). infection, but we observed no significant differences in the number http://www.jimmunol.org/ Although IL-1b, IL-6, and TNF-a are closely associated with of macrophages, monocytes, or DCs at any of the time points disease severity and weight loss, we saw no significant differences measured (Fig. 3, Supplemental Fig. 2B). The significant increases by guest on October 2, 2021

2/2 FIGURE 1. Improved survival of Nlrp12 mice following lethal IAV infection. (A) Mice were infected with a 4LD50 inoculum of IAVand monitored for survival and weight loss through day 14 postinfection. (B and C) Seven days postinfection, IAV-specific CD8+ (B) and CD4+ (C) T cells in the lungs were enumerated using indicated markers. (D) At days 3 and 7 postinfection, virus was quantified in lung homogenate supernatants by plaque assay. (E) H&E- stained sections of lungs harvested 5 d postinfection. Scale bars and original magnification: left, 500 mm(32); middle, 100 mm(310); right, 20 mm(350). Boxes indicate the area magnified in the panel immediately to the right. Black arrows indicate peribronchiolar inflammation, white arrows indicate cellular debris within the airway, solid circle outlines lymphocytic infiltrates, dotted circle outlines scattered mixed inflammatory cells within the pulmonary interstitium. (F) Scoring of necrosis and inflammation in H&E-stained lung sections. (A) Results are representative of two independent experiments, n =10 WT, 9 Nlrp122/2;(B–D) results are representative of two independent experiments, n = 6–8 per group; (E and F) results are representative of one experiment, n = 5 per group, graphed as mean 6 SD. *p , 0.05, one sample t test; **p , 0.01, Gehan–Breslow–Wilcoxon test. 4 Nlrp12 ACTIVATION IS DETRIMENTAL IN INFLUENZA

2/2 FIGURE 2. Nlrp12 mice have decreased pul- Downloaded from monary CXCL1 following lethal IAV infection. (A–F)

Mice were infected with a 4LD50 inoculum of IAVand cytokines and chemokines were quantiﬁed by ELISA on lung homogenate supernatants. Data were pooled from two independent experiments, n = 6–10 per group, error bars represent SEM. Data from individuals with IL-1b values below the limit of detection were not http://www.jimmunol.org/ graphed. All cytokines and chemokines shown were below the limit of detection in lung homogenate supernatants from naive mice. ****p , 0.0001, ordinary one-way ANOVA with Sidak multiple comparisons test. by guest on October 2, 2021

in the frequency of DCs and macrophages at days 5 and 7 postinfection. We further quantified pulmonary neutrophil recruitment infection are due to the decreased total number of hematopoietic in littermate Nlrp12+/+, Nlrp12+/2, and Nlrp122/2 mice following cells, and do not likely represent compensatory recruitment of infection with IAV and found that both Nlrp12+/2 and Nlrp122/2 these cells to the lungs (Supplemental Fig. 2B). Previous studies mice had significantly fewer pulmonary neutrophils than Nlrp12+/+ have shown that IAV accelerates apoptosis in human neutrophils mice (Fig. 4A, 4B), suggesting haploinsufficiency. We also com- (22). We were interested to determine whether a difference in pared neutrophil numbers in the lungs of IAV-infected C57BL/6N survival contributes to the decrease in neutrophils present in the and C57BL/6J sub-strains as C57BL/6J mice carry a missense IAV-infected lungs of Nlrp122/2 mice. We quantified neutrophil mutation in Nlrp12 (13, 23). Consistent with our findings in the death following incubation with IAV, CXCL1, or PMA and found context of bacterial infection, C57BL/6J mice had significantly no increase in death among Nlrp122/2 neutrophils compared with fewer infiltrating pulmonary neutrophils compared with C57BL/ WT neutrophils (Supplemental Fig. 2C). Therefore, increased 6N mice 3 d after IAV infection (Fig. 4C, 4D). Together, these death of neutrophils in the lungs of Nlrp122/2 mice is not likely to results strongly support a role for Nlrp12 in modulating pulmo- be driving the decrease in those cells in the lungs following IAV nary neutrophil accumulation during IAV infection. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

2/2 FIGURE 3. Nlrp12 mice have decreased pulmonary neutrophils during IAV infection. Mice were infected with a 4LD50 inoculum of IAV. Indicated cell populations in lungs were enumerated by ﬂow cytometry at 3, 5, or 7 d postinfection, using gating described in Supplemental Fig. 2A. Three and seven days: pooled from two independent experiments, n = 7–9 per group. Five days: from one experiment, n = 5 per group. Error bars represent SEM. *p , 0.05, , ,

**p 0.01, ***p 0.001, Student t test. by guest on October 2, 2021

Nlrp122/2 mice have decreased vascular permeability during hematopoietic cells than CD45.22 nonhematopoietic cells were IAV infection CXCL1+, and there was no significant difference in the proportion + 2/2 Pulmonary edema due to diminished barrier integrity in IAV-infected of CXCL1 nonhematopoietic cells between WT and Nlrp12 mice (Fig. 6A, 6B). Among immune cell subsets, the frequency of lungs is a serious consequence of both the viral infection and the + resultant immune response, and is associated with increased mor- CXCL1 neutrophils and DCs was significantly decreased in Nlrp122/2 mice, but there was no difference in the proportion of tality (24). Neutrophil-derived reactive oxygen species and cyto- + kines have been implicated in epithelial-endothelial barrier damage CXCL1 macrophages at day 2 or 3 postinfection (Fig. 6C). To during IAV infections (25). The observed decrease in pulmonary determine whether the decrease in CXCL1 was due to a change in neutrophils in Nlrp122/2 mice led us to ask whether there would Cxcl1 transcription, we sorted the same cell populations from IAV- also be less pulmonary vascular permeability in Nlrp122/2 mice infected lungs and measured Cxcl1 transcript levels. We found compared with WT mice. Utilizing Evans blue dye extravasation as decreased Cxcl1 message in DCs and neutrophils at day 2 post- an indicator of vascular permeability we found that at day 5 postinfection, and decreased Cxcl1 message in DCs, neutrophils, and infection Nlrp122/2 mice had significantly decreased vascular macrophages at day 3 postinfection (Fig. 6D). permeability compared with WT mice (Fig. 5A). Nlrp12 is highly expressed in neutrophils, moderately expressed Binding of CXCL1 to CXCR2 on endothelial cells increases in BMDCs, and only modestly expressed in BMMs (16). We microvascular permeability in the lungs (26). When a CXCL1- therefore determined the relative expression of Nlrp12 in CXCL1- blocking Ab was administered to WT mice during IAV infec- producing subsets of cells in the IAV-infected lung. Consistent tion, vascular permeability and pulmonary neutrophils, but not with published data, we found that neutrophils had the greatest 3 DCs or macrophages, were decreased at day 5 postinfection expression of Nlrp12 at days 2 and 3 postinfection, over 10 more 3 (Fig. 5B–E) similar to our findings with Nlrp122/2 mice. There- than in DCs and 100 more than in macrophages (Fig. 6E). To- fore, Nlrp12-dependent modulation of CXCL1 levels plays a role gether, these data demonstrate that both Nlrp12 and Cxcl1 are in vascular permeability during IAV infection. highly expressed in hematopoietic cells. Cellular source of CXCL1 in the lungs during IAV infection Decreased Cxcl1 mRNA stability in Nlrp122/2 BMDCs We next determined the cellular source of CXCL1 in IAV-infected Induction of Cxcl1 can occur downstream of NF-kB activation, lungs. We treated IAV-infected mice with monensin to prevent cells and previous studies have implicated Nlrp12 in the regulation of from secreting CXCL1, then assessed intracellular CXCL1 by NF-kB activation (14, 27, 28). We therefore infected BMDCs with flow cytometry. We found that a higher percentage of CD45.2+ IAVand performed immunoblotting to assess activation of NF-kB 6 Nlrp12 ACTIVATION IS DETRIMENTAL IN INFLUENZA Downloaded from

2/2 +/2

FIGURE 4. Diminished neutrophil recruitment in response to IAV in Nlrp12 , Nlrp12 , and C57BL/6J mice. Mice were infected with a 4LD50 http://www.jimmunol.org/ inoculum of IAV. Indicated cell populations were enumerated in the lungs by flow cytometry at 3 d postinfection. (A and B) Data were pooled from two separate experiments, n = 5–8 per group. (C and D) Data were pooled from two separate experiments, n = 10 per group. Error bars represent SEM. *p , 0.05, **p , 0.01, Student t test. and MAPK family members. Interestingly, we found no pro- Stability of transcripts including Cxcl1, Il6,andTnf can be nounced differences in activation of NF-kB or MAPK family modulated through conserved AU-rich elements (AREs) in their members, despite decreased CXCL1 production by IAV-infected 39 UTRs (30, 31). ARE binding proteins mediate the regulatory Nlrp122/2 BMDCs (Supplemental Fig. 3A–C). These data indi- function of AREs by stabilizing or destabilizing specific mRNAs by guest on October 2, 2021 cated that although the decrease in CXCL1 production by DCs in in response to upstream signaling, commonly from PRRs or IAV-infected lungs is recapitulated in IAV-infected Nlrp122/2 cytokine receptors. Cxcl1 mRNA is present at very low levels in BMDCs, this is unlikely due to alterations in signaling pathways resting myeloid cells, reflecting that the continuous transcription that ultimately converge on Cxcl1 transcription. of Cxcl1 is followed by the binding of these transcripts by the IAV does not robustly infect BMDCs (29), thus it was possible ARE-binding protein TTP, which leads to transcript degradation. that suboptimal or asynchronous stimulation of virus-sensing Upon stimulation of macrophages with LPS, Cxcl1 transcription pattern recognition receptors (PRRs) was obscuring signaling increases and the mRNA is stabilized in part via p38 MAPK and differences. To circumvent this, we treated BMDCs with the MAPKAPK2-mediated phosphorylation of TTP, which affects TLR7 agonist R848 and again observed decreased CXCL1, but its cellular localization and ability to recruit degradation ma- not IL-6, protein, and mRNA in Nlrp122/2 compared with WT chinery to the mRNA (30). Under different stimulatory condi- BMDCs (Fig. 7A–D). Similar to results from IAV infection, we tions, stabilizing (HuR) and destabilizing factors (SF2 via again did not see any differences in activation of NF-kB or MAPK TRAF2/5 and Act1, KSRP) affect the abundance of Cxcl1 family members between WT and Nlrp122/2 BMDCs following mRNA (32). We thus asked if the regulation of Cxcl1 mRNA by challenge with R848, nor did we see any difference in CXCL1 Nlrp12 involved one of these pathways. WT and Nlrp122/2 protein stability (Supplemental Fig. 3D–F). BMDCs were stimulated with R848 followed by immunoblotting Given a lack of evidence for alterations in signaling pathways for ARE-binding and associated proteins. We found no differ- that culminate in transcription of proinflammatory mediators in- ences in the abundance or activation of any of these proteins, nor cluding CXCL1, we next investigated the possibility that Cxcl1 did we see a difference in the activation of translation initiation mRNA stability was different in Nlrp122/2 BMDCs compared factor eIF4E (Supplemental Fig. 4C). Together, these data indi- with WT BMDCs. We measured Cxcl1 and Il6 transcript decay by cate that the mechanism by which Nlrp12 affects Cxcl1 tran- quantitative RT-PCR (qRT-PCR) in R848-treated BMDCs fol- script stability in BMDCs is likely through pathways distinct lowing transcription blockade with ActD. Interestingly, we found from those that have been described downstream of either LPS or an increased rate of Cxcl1 transcript decay in Nlrp122/2 BMDCs cytokine stimulation. compared with WT BMDCs, whereas the rate of Il6 transcript decay was unchanged (Fig. 7E, 7F). The half-life of Cxcl1 Discussion transcript was ∼30 min longer in WT BMDCs than in Nlrp122/2 This work demonstrates that Nlrp12 contributes to disease se- BMDCs, but no difference in Cxcl1 transcript stability was ob- verity during IAV infection through its effect on CXCL1 pro- served in BMMs or neutrophils (Supplemental Fig. 4A, 4B). To- duction, neutrophil recruitment to the lungs, and vascular gether, these data suggest that Nlrp12 plays a role in Cxcl1 permeability. Our finding that Nlrp122/2 mice have decreased— transcript stability specifically in BMDCs. but not ablated—pulmonary neutrophils and improved survival is The Journal of Immunology 7

FIGURE 5. Nlrp122/2 mice have decreased pulmonary microvascular permeability. (A) Mice were infected with a 4LD50 inoculum of IAV. At day 5 postinfection, vascular permeability was measured by Downloaded from Evans blue extravasation. (B–E) On days 0 and 3 of infection, mice were administered CXCL1-blocking Ab or isotype control Ab i.p. (B) At day 5 postinfection, vascular permeability was measured by Evans blue extravasation. (C–E) Indicated cell populations were enumerated by ﬂow cytometry. Data were pooled from two independent experiments, http://www.jimmunol.org/ n = 8–11 per group. Error bars represent SEM. **p , 0.01, Student t test. by guest on October 2, 2021

important because it supports a protective effect of neutrophils barrier in the alveolus, which allows accumulation of proteina- during IAV infection that is dependent on the magnitude of the ceous fluid and immune cells that dramatically impair efficient gas neutrophilic response. We report a 25–40% decrease in the exchange. CXCL1 can affect the barrier in at least two ways: first, number of pulmonary neutrophils during infection, and show by binding to CXCR2 on endothelial cells, causing retraction of that this curtails enough of the immunopathology driven by the endothelial cells to increase microvascular permeability (26); excessive innate inflammatory responses without negatively af- second, by attracting CXCR2-expressing neutrophils to the site of fecting viral clearance or CD8+ T cell responses, to result in infection, where they have the capacity to directly damage the improved survival. We also show that treatment with CXCL1- epithelial cells in the lungs in the course of fighting infection (11). blocking Ab results in decreased neutrophil recruitment and Thus, the decrease in CXCL1 observed in Nlrp122/2 animals may decreased vascular permeability, consistent with the notion that be protective to both sides of this critical barrier in the lung, ul- downmodulation of early inflammatory mediators decreases timately lessening disease severity. disease severity. An important avenue of study will be to determine the mech- The specific decrease in CXCL1 has broad implications for the anism by which Nlrp12 deficiency affects CXCL1 production. A overall course of disease due to direct and indirect effects on barrier recent study reported that CXCL1, CXCL5, IL-17A, and neutrophil function in the IAV-infected lung. The main complication of IAV recruitment were decreased in Nlrp122/2 mice during pulmonary infection is viral pneumonia, which can lead to acute respiratory Klebsiella pneumoniae infection (15). Signaling through IL-17R distress syndrome, respiratory failure, and death (24). Respiratory leads to stabilization of Cxcl1 and Cxcl5 mRNA (32), which may failure is precipitated by damage to the epithelial-endothelial explain the decrease observed during K. pneumoniae infection. 8 Nlrp12 ACTIVATION IS DETRIMENTAL IN INFLUENZA Downloaded from

FIGURE 6. Decreased CXCL1 production by immune cells in the lungs of Nlrp122/2 mice during

IAV infection. Mice were infected with a 4LD50 inoculum of IAV. (A–C) At the indicated day postin- http://www.jimmunol.org/ fection, CXCL1+ cell populations were enumerated in the lungs by ﬂow cytometry. (D and E) At the indicated day postinfection, cell populations were puri- ﬁed by FACS and gene expression was determined by qRT-PCR. (A–C) Results are representative of two independent experiments, n = 3–5 per group, error bars represent SEM. (D and E) Data were pooled from two independent experiments, graphed as mean 6 SEM *p , 0.05, **p , 0.01, ***p , 0.001, Student by guest on October 2, 2021 t test.

However, IL-17A was not detected in the lungs of WT or Nlrp122/2 similar patterns of transcriptional regulation. It is well established mice during IAV infection, thus it is unlikely to be driving the that modulation of neutrophil chemoattractant transcript stability observed decrease in Cxcl1 transcript stability. It is particularly is an important determinant of both the duration and magnitude of intriguing that we observe a defect in CXCL1, and not other their expression, and that ARE-mediated instability is a key reg- neutrophil chemoattractants or proinﬂammatory mediators with ulatory mechanism. Despite this generality, it is also clear that the The Journal of Immunology 9

FIGURE 7. Decreased Cxcl1 transcript stability in R848-stimulated Nlrp122/2 BMDCs. (A–D) BMDCs were treated with 1 mg/ml R848 for the indicated amount of time and CXCL1 and IL-6 gene expression and protein were quantiﬁed. (E and F) Downloaded from BMDCs were treated with 1 mg/ml R848 for 3 h followed by the addition of ActD. At the indicated times after addition of ActD, RNA was extracted and qRT-PCR was used to quantify remaining Cxcl1 or Il6 transcripts. Results are presented as the percentage of transcript present immediately prior http://www.jimmunol.org/ to ActD treatment (0 h). (A–D) Data shown are representative of two independent experiments. (E and F) Data were pooled from three separate experiments. All data are graphed as mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, ordinary one-way ANOVA (A–D), two-way repeated measures ANOVA (E and F) with Sidak multiple comparisons test (A–F). by guest on October 2, 2021

precise regulation of the production of the chemokine reflects not The importance of host genetic background to outcome during only which gene is being regulated, but also varies according to IAV infection has been demonstrated in a number of studies (34). the cell type in which the gene is being expressed as well as by the Large-scale efforts such as the collaborative cross that seeks to specific conditions under which that cell is being stimulated. mimic the genetic diversity found in the human population are Differential regulation of TNF-a is illustrative of this mechanistic validating the importance of specific genes and discovering new heterogeneity: Tnf mRNA is stabilized following LPS stimulation correlates of protection (35). To our knowledge, there has not been of macrophages, resulting in increased expression, but following a direct comparison of C57BL/6N and C57BL/6J substrains dur- stimulation of macrophages through TLR7 or TLR9, upregulation ing IAV infection. The data presented in this study indicate that is not through modification of transcript stability but rather occurs there is a significant reduction in the number of pulmonary neu- at the level of translation (33). Given this highly variant regulation trophils in the lungs of C57BL/6J mice compared with C57BL/6N and the fact that most studies of Cxcl1 stability in myeloid cells mice following IAV infection. In the context of these data and our have been performed in LPS-treated macrophages, it is not previous work, we believe this difference in neutrophil recruit- unexpected that a separate pathway would act to specifically ment is due to the missense mutation in Nlrp12 in C57BL/6J mice modify Cxcl1 transcript stability in BMDCs in response to R848 (13, 23), which phenocopies the neutrophil recruitment defect in stimulation. Nlrp122/2 mice. This substantial phenotypic difference between 10 Nlrp12 ACTIVATION IS DETRIMENTAL IN INFLUENZA substrains highlights the importance of ensuring that findings in causes C57BL/6J strain-specific defect in neutrophil recruitment. Nat. Commun. 7: 13180. knockout mice are confirmed using substrain-matched controls or 14. Allen, I. C., J. E. Wilson, M. Schneider, J. D. Lich, R. A. Roberts, J. C. Arthur, littermates when possible. R. M. Woodford, B. K. Davis, J. M. Uronis, H. H. Herfarth, et al. 2012. NLRP12 In conclusion, the experiments described in this study show that suppresses colon inflammation and tumorigenesis through the negative regula- 2/2 tion of noncanonical NF-kB signaling. Immunity 36: 742–754. Nlrp12 mice are protected from lethality during IAV infection, 15. Cai, S., S. Batra, F. Del Piero, and S. Jeyaseelan. 2016. NLRP12 modulates host exhibiting less pulmonary microvascular permeability, CXCL1, defense through IL-17A-CXCL1 axis. Mucosal Immunol. 9: 503–514. and neutrophil recruitment. Modulation of the innate immune 16. Arthur, J. C., J. D. Lich, Z. Ye, I. C. Allen, D. Gris, J. E. Wilson, M. Schneider, K. E. Roney, B. P. O’Connor, C. B. Moore, et al. 2010. Cutting edge: NLRP12 response to IAV is being increasingly appreciated as a key early controls dendritic and myeloid cell migration to affect contact hypersensitivity. intervention with durable protective effects. Our findings add J. Immunol. 185: 4515–4519. 17. Legge, K. L., and T. J. Braciale. 2005. Lymph node dendritic cells control CD8+ Nlrp12 to the intracellular PRRs that contribute to early inflam- T cell responses through regulated FasL expression. Immunity 23: 649–659. matory responses during IAV infection. 18. Anderson, K. G., K. Mayer-Barber, H. Sung, L. Beura, B. R. James, J. J. Taylor, L. Qunaj, T. S. Griffith, V. Vezys, D. L. Barber, and D. Masopust. 2014. Intra- vascular staining for discrimination of vascular and tissue leukocytes. Nat. Acknowledgments Protoc. 9: 209–222. We are grateful to members of the Inflammation Program, Eric Elliott, 19. Moitra, J., S. Sammani, and J. G. Garcia. 2007. Re-evaluation of Evans Blue dye Bruce Hostager, Nurbek Mambetsariev, Alexis Miller, Diogo Valadares, as a marker of albumin clearance in murine models of acute lung injury. Transl. Res. 150: 253–265. and Alicia Wallis for valuable discussions and technical assistance. We 20. Helft, J., J. Bo¨ttcher, P. Chakravarty, S. Zelenay, J. Huotari, B. U. Schraml, thank Tamara Mirzapoiazova for generosity with microvascular permeability- D. Goubau, and C. Reis e Sousa. 2015. GM-CSF mouse bone marrow cultures related protocols and the University of Iowa Flow Cytometry Facility, a comprise a heterogeneous population of CD11c(+)MHCII(+) macrophages and Carver College of Medicine/Holden Comprehensive Cancer Center core re- dendritic cells. Immunity 42: 1197–1211. 21. McDermott, D. S., and S. M. Varga. 2011. Quantifying antigen-specific CD4 search facility, as well as the University of Iowa Comparative Pathology T cells during a viral infection: CD4 T cell responses are larger than we think. Downloaded from Laboratory, for expertise. J. Immunol. 187: 5568–5576. 22. Colamussi, M. L., M. R. White, E. Crouch, and K. L. Hartshorn. 1999. Influenza A virus accelerates neutrophil apoptosis and markedly potentiates apoptotic ef- Disclosures fects of bacteria. Blood 93: 2395–2403. The authors have no financial conflicts of interest. 23. Simon, M. M., S. Greenaway, J. K. White, H. Fuchs, V. Gailus-Durner, S. Wells, T. Sorg, K. Wong, E. Bedu, E. J. Cartwright, et al. 2013. A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains. Ge-

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